NEURONAL TRANSFORMATION BY INDUCING NEURON-SPECIFIC TRANSCRIPTION FACTORS.

本论文对神经元特异性和变异性的分子机制进行了研究，以期在神经退行性疾病的治疗中有所应用。在第一步，我们集中在转录调控的D1 A多巴胺受体基因作为一个代表性的分子，其特征的一个子集的神经元和POU转录因子基因家族广泛表达，但具体在中枢神经系统。将D1 A受体基因的上游基因组片段亚克隆到CAT报告质粒中，通过PCR将其缺失至不同长度，并用于顺式元件的分析。我们共转染POU基因家族成员Brn-4，并观察其转录激活。通过该分析，在D1 A受体基因的第一内含子中发现了Brn-4反应元件，其包含两个用于结合POU因子的共有序列。用重组GST-Brn-4蛋白进行凝胶迁移率变动分析证实， ...更多信息 这些基序是scrual结合位点。然后我们通过原位杂交测试Brn-4和D1 A是否共定位于纹状体神经元中，并发现这两种分子存在于类似的神经元组中。这些结果表明，Brn-4实际上在体内作用于其D1 A多巴胺受体基因的响应元件，从而影响其转录调控。此外，我们发现POU家族成员对相同的元素有不同的作用。Oct 2和Brn-4正调节D1 A基因的转录，而Brn-2、Oct-3和Oct-6则不调节。当Oct-6与Brn-4共转染时，其竞争性抑制Brn-4的反式激活。这些结果可能提示某些共转录因子与特定的POU成员结合并调节其反式激活。我们还发现，在D1 A阴性神经元细胞系中，一些抑制转录因子作用于D1 A受体基因的上游。我们正在尝试克隆未知的因子，今年获得的这些结果表明POU转录因子与D1 A受体（一种定义神经元特异性的神经元基因）之间的分子关系。下一步，我们希望通过抑制转录因子或其他实验方法来调控神经元的特异性。少

We have investigated the molecular mechanism controlling specifity and variability of neurons in expect of its application for the therapy of neurodegenerative disorders in the future. At the first step, we focused on transcriptional regulation of the D1A dopamine receptor gene as a representative molecule which characterixes a subset of neurons and on the POU transcription factor gene family that are expressed widely but specifically in the central nervous system. The upstream genomic fragment of the D1A receptor gene was subcloned into a CAT reporter plasmid, deleted to various length by using PCR,and used for the analyzes on cis-element. We co-transfected Brn-4, a member of the POU gene family, and observed its transcriptional activation. Through this assay, a Brn-4 responsive element was found in the first intron of the D1A receptor gene which contained two consensus sequences for binding of POU factors. Gel mobility shift assays with recombinant GST-Brn-4 protein confirmed that th … More ese motifs were scrual binding sites. We then tested whether Brn-4 and D1A colocalizes in the striatal neurons by in situ hybridization, and found that these two molecules exist in a similar group of neurons. These results suggest that Brn-4 actually acts on its responsive element of the D1A dopamine receptor gene in vivo, and thereby influence on its transcriptional regulation. Furthermore, we found that POU family members differentially act on the same element. Oct2 and Brn-4 positively regulate transcription of the D1A gene, whereas Brn-2 Oct-3 and Oct-6 not. When Oct-6 was co-transfected with Brn-4, it copetitively inhibited transactivation by Brn-4. These results may suggest that some co-transcription factors which bind to specifiic POU members and modify their transactivation. We also found that, in a D1A-negative neuronal cell line, some repressing transcription factors act on the upstream of the D1A receptor gene. We are now trying to clone the unknown factors.These results obtained in this year showed a molecular relationship between POU transcription factors and D1A receptor, a neuronal gene which defines the specificity of neruons. In the next step, we wish to regulate the specific character of neurons by transfecting transcription factors or by other experimental approaches. Less